Field of the Invention and Related Art Statements
The present invention relates to a method of measuring a specific binding reaction with the aid of a polarized light beam and a magnetic field.
There has been developed an immunological analysis for measuring immune substances, hormones, medicines, and various components such as immune regulators faintly contained in living bodies by utilizing a specific immunological reaction. It should be noted that in the present specification, the term specific binding reaction is used to express all reactions due to ligands which are reactive to specific substances. Therefore, the specific binding reaction includes not only the immunological reaction due to antigen-antibody reaction, but also the avidin-biotin reaction, protein A-IgG Fc fragment reaction and hormone-receptor reaction. For instance, the immunological analysis may be roughly classified into a labeling immunological analysis in which enzymes and isotopes are used as an indicator substance, and a non-labeling immunological analysis in which antigen-antibody complexes are directly measured.
In prior labeling immunological analysis, there have been widely known radio immuno assay (RIA), enzyme immuno assay (EIA) and fluoro immuno assay (FIA). These assays have an advantage in that a high sensitivity can be attained, but also have a drawback in that handling of isotopes and wasted liquid is difficult, measuring periods are long and labeling reagents are expensive so that the test cost, per sample, i.e. running cost is often high.
In prior non-labeling immunological analysis, there have been developed immuno electrophoresis, immuno diffusion and sedimentation. These methods have been described in detail in Japanese magazines, "Summary of Clinical Test Method" published by Kinbara and "Clinical Test", vol 22, No. 5 (1978) pp. 471-487.
In "Immuno chemistry", vol 12, No. 4 (1975) pages 349 to 351, there has been proposed one method of non-labeling immunological analysis in which an antigen or antibody bound on surfaces of fine particles is reacted with an antibody or antigen contained in a test liquid, and an average diffusion constant which is an indicia of the Brownian motion of aggregates composed of agglutinated particles is measured from a variation in a spectral width of laser light scattered from a solution of particles. This method has a merit that no reagent is used. However, since the spread of the spectrum due to the Doppler effect owing to the Brownian motion of aggregates is detected by a spectrometer, the apparatus is liable to be large in size and expensive in cost. Further, error might be induced when the spectrometer is driven mechanically, so that precision and reproducibility become worse. Moreover, in this known method in which the average diffusion constant is measured from the spectral width, the amount of available information about the antigen-antibody reaction is limited.
The present inventor has proposed, in U.S. patent application Ser. No. 754,272 filed on July 12, 1985, a method of measuring an antigen-antibody reaction, in which it is not necessary to use expensive reagents or a sepectrometer, the measurement can be carried out reproducibly at a high precision, and the measurement can be performed automatically within a relatively short time period.
This method of measuring an immunological reaction comprises the steps of:
projecting radiation into a reaction liquid containing at least antigen and antibody;
detecting radiation scattered by particulate substances in the reaction liquid;
deriving a power spectrum density of fluctuation in intensity of said scattered radiation; and
measuring an antigen-antibody reaction on the basis of said power spectrum density.
After conducting various experiments and analyses, the inventor has found that if impurities such as highly polymerized substances are contained in a test liquid, the light beam is also scattered by the impurities, and thus an output signal supplied from a photodetector includes error components due to said highly polymerized substances.